The present invention relates to biosensors with porous membranes comprising
(a) at least one substrate;
(b) an electrode layer patterned on the substrate, consisting of an electrode system and a circuit connector;
(c) an insulator, for separating the electrode system and a circuit connector formed on parts of the electrode layer;
(d) a porous membrane covered with the surface of the electrode system by the insulator; and
(e) a protective membrane for protecting the porous membrane, formed on the porous membrane, or upper substrate containing a sample inlet for protecting the porous membrane as well as being introducible samples wherein, when a whole blood sample is introduced to the biosensor, the whole blood sample is separated into its components during the chromatographic motion through the porous membrane so that only blood plasma can be contacted with the electrode system.
Periodical monitoring of blood glucose levels is needed for the diagnosis and prophylaxis of diabetes mellitus. Adopting colorimetry or electrochemistry as their operation principle, strip type analyzers are conventionally used to determine glucose levels in blood.
Such a calorimetric principle is based on the glucose oxidase-colorimetric reaction represented by the following reaction formula 1:
Glucose+O2xe2x86x92Gluconic acid+H2O2 (Glucose Oxidase Catalysis)
H2O2+Oxygen Receptorxe2x86x92(oxidized) Oxygen Receptor+2H2O (Peroxidase Catalysis)xe2x80x83xe2x80x83Reaction Formula 1
In the presence of oxygen, as illustrated in the above formulas, glucose is oxidized with the aid of glucose oxidase to produce gluconic acid and hydrogen peroxide. From the hydrogen peroxide, oxygen molecules are transferred to an oxygen receptor (chromophore) by the catalysis of peroxidase. As a result of the oxidation, the chromophore changes color, and its color intensity is the basis of the quantitative analysis of blood glucose levels.
In order to utilize this calorimetric principle, however, precise care must be taken as to sample transport, pre-treatment, quantity, reaction time, and coloration starting time. In addition, blood coagulation or various interfering materials, including uric acid, ascorbic acid and bilirubin, may disturb the calorimetric analysis. Furthermore, the accompanying photometry has the fundamental limitation that its analytical accuracy and precision is lowered at high and low concentrations of samples. With these problems, the calorimetric analysis is known to be inappropriate for accurate quantification.
To avoid the problems that the calorimetric analysis has, electroanalytical methods were chemistry was introduced to biosensors. Over the calorimetric biosensors, the electrochemical biosensors have the advantage of being higher in selectivity and sensitivity, being able to measure the colored or turbid samples, without pre-treatment, and being able to perform accurate analysis within a short period of time. In order to better understand the background of the invention, the electroanalytical chemistry on which the second-generation biosensor will be described in conjunction with the following formula 2 and FIG. 1. In contrast to the first-generation biosensor which uses oxygen as an electron transfer mediator, the second-generation biosensor takes advantage of an electron transfer mediator selected from the group comprising ferrocene, ferrocene derivatives, quinones, quinone derivatives, organic conducting salts, and viologen.
xe2x80x83Glucose+GOX-FADxe2x86x92Gluconic acid+GOX-FADH2
GOX-FADH2+Electron Transfer Mediator (Oxidized)xe2x86x92GOX-FAD+Electron Transfer Mediator (Reduced)xe2x80x83xe2x80x83Reaction Formula 2
In Reaction Formula 2, GOX represents glucose oxidase, and GOX-FAD and GOX-FADH2 are the oxidized and reduced forms of the glucose oxidase, respectively, because FAD (flavin adenine dinucleotide) is the active site of glucose oxidase. In FIG. 6, there is an electron transfer system for the electrochemical analysis of blood glucose levels, in which glucose oxidase and ruthenium are used as electron carriers from glucose to an electrode. Glucose is oxidized to gluconic acid by the catalytic action of glucose oxidase while the active site, FAD, of the glucose oxidase is reduced to FADH2, which transfers its electron, in turn, to the electron transfer mediator while being returned to the oxidized form FAD. The reduced electron transfer mediator ruthenium is diffused to the surface of an electrode. At the surface of the electrode, measured is the current generated when the oxidation potential of the reduced electron transfer mediator is applied. The oxidation potential is in the range of xe2x88x920.2 to 0.2 V versus a reference electrode, so that the influence of ascorbic acid and uric acid, which have oxidation potentials higher than 0.3 V and 0.4 V, respectively, can be excluded.
In contrast to the first-generation biosensor, therefore, the second-generation biosensor is not affected by oxygen. In addition, the second-generation biosensor enables the non-selective catalysis of an oxidase and uses an electron transfer mediator, which has such appropriate redox potentials as to reduce the error caused by interfering materials, so that the measured potentials can be used to accurately determine the analytical quantity of interest. However, problems are also found in the second-generation biosensor. Because the electron transfer mediator, after the electron transfer to and from the oxidase, must diffuse to the surface of the electrode to detect the electrochemical change, a large quantity of the electron transfer mediator is needed. The abundance of the electron transfer mediator may alter the three-dimensional structure of the oxidase, resulting in a decrease in the activity of the enzyme. Another problem with most electron transfer mediators is high reactivity with electrode-activating materials in the blood.
Most of the glucose sensors, which are commercially available to date, follow the principle of the second-generation biosensors. Since the electron transfer mediators employed in most commercially available glucose sensors to be oxidized at potentials similar to oxidation potentials of the interfering materials within blood, such as ascorbic acid, acetaminophene and uric acid, the influence of the interfering materials within blood is not completely eliminated. To circumvent these problems, some commercially available glucose sensors take advantage of capillarity in introducing blood samples thereinto, but suffer from the disadvantage that their fabrication is complicated because there is required the process of coating hydrophilic polymers onto hydrophobic supports to achieve the introduction.
Recently, intense attention has been paid to the use of immunochromatographic methods in biosensors. In an immunochromatographic biosensor, a porous membrane is provided to form an electrode in its upper part and a sample pretreatment layer in its lower part. Without additional operations for pretreatment, the sample was moved through chromatographic action, during which a target material of the sample can be quantitatively determined by the change in electrical quantity of the target material (Cui, G., Kim, S. J., Choi, S. H., Nam, H., Cha, G. S, and Paeng, K. J., Anal. Chem., 2000, 72, 1925-1929; Cha, G. S. et al., U.S. App. Ser. No. 09/381788 now U.S. Pat. No. 6,210,907; Farm, M. L., Rorad, O. H., Park, H., International Pat. WO 00/00827 2000). It is, however, difficult to introduce an electrode and a pretreatment layer together to a porous membrane for chromatographic motion. To relieve the difficulty, an electrode is formed on a plastic substrate while a porous membrane is used for a sample introducing part. This technique, however, is disadvantageous in that the electrode does not adhere well to the porous membrane of the sample introducing part that the sensor is poor in reproducibility. Additionally, it takes a long period of time for a sample to travel through the pretreatment layer to the sensor such that the immunochromatographic biosensor, which composed of a sample pretreatment layer in its lower part, suffers from the disadvantage of requiring a long time for analysis.
To circumvent the problems encountered above, the present inventors offered a biosensor having a structure in which a porous membrane capable of chromatographically separating blood cells and blood plasma is positioned on an electrode while being pressurized against the electrode by a cover (KR 10-2000-041962). This biosensor is advantageous in that a ruthenium based electron transfer mediator is deposited on the porous membrane to lower the oxidation potential, thereby reducing the error attributable to interfering material. Also, this porous membrane-built-in biosensor enjoys the advantage of significantly reducing errors attributable to blood cell/blood plasma ratios and to easily oxidized materials. Another advantage of this biosensor resides in its structure, which has immobilized enzymes and electrode-active materials intercalated between two closed boards and thus isolated from external air, so that their lifetime can be prolonged without using separate packages. However, a serious problem with this biosensor is to make it difficult to introduce viscous blood samples thereinto.
Leading to the present invention, the intensive and thorough research on biosensors, conducted by the present inventors, a biosensor comprises: (a) at least one substrate; (b) an electrode system, formed on one end of the substrate, having a sensing material; (c) an insulator, formed on parts of the electrode layer, for electrically separating the electrode system from a circuit connector; (d) a porous membrane, covered with the surface of the electrode system, having an oxidase and an electron transfer mediator; and (e) a protective membrane as large as the porous membrane and/or an upper substrate equipped with a sample inlet, are laminated in sequence, can filter samples chromatographically through the pores of the porous membrane, be preserved for a long period of time because the sensor materials and porous membrane are closed by the upper substrate, and conduct maximum performance without being disturbed by interfering components of samples. In addition, it is found that the biosensor structure can introduce samples at constant rates to the electrodes without pretreatment, to quantify levels of samples rapidly, and to sense changes in sample level with high consistency. Further to these, the biosensors are found to allow patients to detect blood levels of metabolites associated with various diseases accurately irrespective of interfering materials and by themselves.
Therefore, it is an object of the present invention to provide a biosensor which composes: (a) at least one substrate; (b) an electrode system, formed on one end of the substrate, having a sensing material; (c) an insulator, formed on parts of the electrode layer, for electrically separating the electrode system from a circuit connector; (d) a porous membrane, formed on the electrode system, having an oxidase and an electron transfer mediator; and (e) a protective membrane for protecting the porous membrane.
It is another object of the present invention to provide a biosensor composing: (a) a substrate; (b) an electrode system, formed on one end of the substrate, having a sensing material; (c) an insulator, formed on parts of the electrode layer, for electrically separating the electrode system from a circuit connector; (d) a porous membrane, formed on the electrode system, having an oxidase and an electron transfer mediator; (e) a second electrode pattern formed on the porous membrane, facing the electrode pattern; and (f) an upper substrate, formed on the adhesive, for protecting the biosensor, having a hole through which samples are introduced inside the biosensor
It is another object of the present invention to provide a biosensor with a modified sample inlet capable of rapidly introducing a predetermined amount of blood samples and increasing accuracy and reproducibility.